Functional characterization of the efflux pump OqxAB by complementation assays in E. coli Purnendu Bhowmik, Anirudh P Shanbhag, Nagakumar Bharatham and Nainesh Katagihallimath BUGWORKS Research India Pvt. Ltd., C-CAMP, NCBS-TIFR, GKVK Campus, Bellary Road, Bangalore 560065, India ABSTRACT Minimum Inhibitory Concentration (MIC) Assays Nile Red Assay

Efflux of antibiotics is a major mechanism of microbial resistance and is primarily The RND pump components from E. coli, Pseudomonas aeruginosa and Klebsiella Nile Red is a membrane localising cytostain that can be used to study substrate efflux and mediated by the AcrAB-TolC complex and its homologs. In the spectrum of homologs, pneumoniae were studied by cloning and expressing them in E. coli ∆acrB/∆acrAB deficient their inhibition or competition in whole E. coli cells. The efflux efficiencies of the E. coli

OqxAB from Klebsiella pneumoniae is phylogenetically distant from the other members of systems. MexB, OqxB, MexAB and OqxAB formed functional complexes in the heterologous ∆acrB/∆acrAB and their complemented strains are compared using their tefflux50% for Nile the RND efflux pumps across bacteria and is the least studied and characterized, unlike the system to efflux antibiotics like Linezolid, Novobiocin and Erythromycin in MIC experiments. Red in the presence and absence of the competing antibiotics. E. coli counterpart acrAB. The genes oqxAB confer antibacterial resistance not only in K. pneumoniae but also in other organisms like E. coli, Salmonella, and Enterobacter Complementation in ∆acrB aerogenes. The dissemination of this new efflux-based drug resistance in Gram-negative bacteria by either its association with the insertion element IS26 or plasmid-borne MIC Values (μg/ml) ∆acrB complementation 24 STRAINS LZD NOV ERY RIF transmission is a new and alarming threat. The present study focuses on the LZD NOV ERY RIF C43(DE3) (WT) 160 80 80 5 complementation of OqxAB in E. coli efflux pump knockout strains by Minimum Inhibitory C43(DE3)-∆acrB 10 5 5 5 20 ∆acrB+pET21a 5 10 10 2.5 Concentration and Nile Red efflux assays. In-silico analysis suggests that the efflux of ∆acrB+p_acrB 80 40 80 5 compounds by OqxB is similar to AcrB and MexB despite being phylogenetically distant. Our ∆acrB+p_mexB 80 40 40 5 16 16 16 studies give an insight into the efflux capabilities of this pump system against various efflux ∆acrB+p_oqxB 40 40 40 2.5 liable antibiotics in wild type E. coli, and efflux pump deleted strains. In-silico Studies 10 10 10 8

4

Fold Change in MIC w.r.t. ∆acrB MIC in w.r.t. Change Fold 2 2 1.3 0.83 1 0.83 1 1 1 1 1 1 1 0.83

C43(DE3)-WT ∆ACRB+P_ACRB ∆ACRB+P_MEXB ∆ACRB+P_OQXB C43(DE3)_∆ACRB ∆ACRB + PET21A

Complementation in ∆acrAB

24 MIC Values (in μg/ml) for ∆acrAB complementation LZD NOV ERY RIF E. coli Strains LZD NOV ERY RIF Distance based phylogenetic tree of OqxA and its homologs in bacterial pathogens shows BW25113 160 80 80 5 that OqxA and AcrA share little sequence identity. Similarly OqxB and AcrB are ∆acrAB (∆2) 5 10 5 5

acrAB ∆acrAB+ pTrc99a 10 5 5 5 ∆ phylogenetically distant from each other. 16 ∆acrAB+ p_acrAB 40 40 10 2.5

∆acrAB+ p_mexAB 40 80 20 5 w.r.t. ∆acrAB+ p_oqxAB 80 40 10 5

10 10

8

6 5

4 4 4 Fold Change MIC in Change Fold 2 2 1.3 0.6 0.8 0.6 1 1 1 1.0 1 1 1 0.8

BW25113-WT ∆ACRAB+P_ACRAB ∆ACRAB+P_MEXAB ∆ACRAB+P_OQXAB ∆ACRAB ∆ACRAB+PTRC99A

A B C

AcrB Substrate binding region OqxB Substrate binding region LINEZOLID

Efflux of Nile Red in the wildtype, lack of it in the ∆acrB/∆acrAB strains and their complementation is shown in Figures A and B. Figures C - J represent the efflux of Nile Red D E F at saturating concentrations of Linezolid, Erythromycin, Ciprofloxacin and Novobiocin.

3 0 0 3 0 0

Superimposition of AcrB & Superimposition of AcrB & C 4 3 ( D E 3 ) 6 5 3 0 0 2 6 0 1 4 0 3 0 0 B W 2 5 1 1 3 4 4 2 0 3 2 1 4 1 6 5 3 0 0 MexB cage residues OqxB cage residues

NOVOBIOCIN  a c r B 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0  a c r A B 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 2 0 0 2 0 0

Protein Distal binding pocket residues p _ a c r B 7 0 1 5 0 1 6 9 1 9 2 2 7 5 p _ a c r A B 6 0 1 2 1 1 4 6 1 4 0 1 2 0 AcrB F136 F178 I277 F610 V612 F615 F617 F628 MexB F136 F178 I277 F610 V612 F615 F617 F628 p _ m e x B 4 5 2 2 1 2 1 5 1 7 1 2 3 0 p _ m e x A B 5 5 2 1 5 2 1 0 1 3 5 1 7 2 OqxB L138 F180 L280 V616 F618 L621 A623 F636 G H I 1 0 0 1 0 0 p _ o q x B 7 2 1 5 5 1 6 8 1 4 5 1 9 6 p _ o q x A B 6 8 1 7 8 1 9 5 1 4 5 1 5 8

C T R L L Z D C I P E R Y N O V C T R L L Z D C I P E R Y N O V

Heat map of Nile Red tefflux50% in wild type, ΔacrB, ΔacrAB and their respective complementation with overexpressed heterologous counterparts; MexB, OqxB, MexAB and

OqxAB. The knockouts (ΔacrAB and C43 (DE3)-ΔacrB) completely inhibit the Nile Red efflux. ERYTHROMYCIN

In the presence of antibiotics, the tefflux50% for Nile Red increases due to competition as Determination of MIC on agar plates to check for complementation by overexpressing depicted in shades of red. The map shows that the overexpression of cognate A and B AcrB MexB OqxB acrB, mexB and oqxB in ∆acrB background. Different concentrations of the compounds like proteins is more efficient than B alone to efflux the molecules. AcrA AcrA & MexA AcrA & OqxA superimposed superimposed Linezolid (LZD) : Plates A-C; Novobiocin (NOV): Plates D-F and Erythromycin (ERY) : Plates G-I, were tested to check the sensitivity of the strains to complement the loss of acrB. The substrate binding regions (phenylalanine cage) of AcrB and MexB are identical while the Conclusion OqxB has similar residues. The overall structure fold remains similar in all of them. AcrA and Acknowledgements K. pneumoniae OqxB can form a chimeric functional complex with E. coli AcrA to efflux OqxA have an identity of only 28% but our homology model shows that they are structurally antibiotics. Despite having a low protein sequence homology, AcrB & OqxB and AcrA & similar. We evaluated if the chimeric complex of AcrA and OqxB could efflux molecules in a This study is supported by BIRAC, DBT, Govt. of India (BT/BIPP/0803/30/14, C- OqxA have high structural similarity, thus able to complement each other as evidenced by Minimum Inhibitory Concentration experiment and Nile Red Efflux Assay. CAMP/BIG/98B) and CARB-X (6 IDSEP160030-01-02). the in-silico modeling, MIC and Nile Red efflux assay.